Summary Hepatitis E virus (HEV), an enterically transmitted virus, exists either as naked virions that are shed in the feces or quasi-enveloped virions that circulate in the blood. The quasi-envelopment of HEV facilitates noncytolytic release of virus from infected cells. However, the lack of viral envelope proteins raises question about mechanism for HEV spread and the role of antibodies in natural infection. HEV infects ~20 million people annually and causes significant morbidity and mortality. Persistent HEV infection in immunocompromised individuals can lead to rapid progression of liver fibrosis. Currently there are no FDA-approved diagnostics and HEV-specific therapies. Our long-term goal is to unravel the functional role(s) of this unusual envelopment in the virus life cycle and pathogenesis for the purpose of identifying novel targets for therapeutic intervention. The overall objective of this project is to understand the role of the quasi-envelopment in HEV spread and how it affects antibody-mediated neutralization. The viral ORF3 protein is known to play a key role in HEV envelopment. Our preliminary data suggest that ORF3 is subjected to regulation by palmitoylation to mediate virion release. In addition, we found evidence that neutralizing antibodies block HEV spread in cells that have already established infection. Our central hypothesis is that ORF3-mediated quasi-envelopment is required for HEV release into the bloodstream and into the bile (then shed into feces), and quasi-enveloped HEV particles mediate spread with the liver via a novel entry mechanism that is susceptible to post-entry neutralization by antibodies. We will test this hypothesis in polarized hepatocyte cell culture and in human liver chimeric mice. Aim 1 will define the role of palmitoylation of ORF3 in its cellular localization and function in virus release. Aim 2 will define the cellular requirements and role of ORF3 in HEV spread. Aim 2 will also define the role and mechanism of antibodies in blocking HEV spread. The expected outcomes of the proposed research are novel insights into the spread mechanism for a quasi- enveloped virus, and may have implications for other non-enveloped viruses such as hepatitis A, poliovirus, and enteroviruses that also become quasi-enveloped at certain stages of their life cycle. This work will fill gaps in our understanding of the quasi-envelopment processes and how it influences virus spread, pathogenesis and immunity.